1.1 Field of The Invention
The invention is in the field of molecular biology. More specifically, the invention relates to methods for the large-scale production of recombinant adeno-associated virus (rAAV) for use in gene therapy applications.
1.2 Description of The Related Art
Gene therapy refers to treatment of genetic diseases by replacing, altering, or supplementing a gene responsible for the disease. It is achieved by introduction of a corrective gene or genes into a host cell, generally by means of a vehicle or vector. Gene therapy holds great promise for the treatment of many diseases. Already, some success has been achieved pre-clinically, using recombinant AAV (rAAV) for the delivery and long-term expression of introduced genes into cells in animals, including clinically important non-dividing cells of the brain, liver, skeletal muscle and lung. Clinical trials using this technology have included use of rAAV expressing the cftr gene as a treatment for cystic fibrosis (Flotte et al., 1998; Wagner et al. 1998).
Methods for production of rAAV have been developed in which cells grown in culture are caused to produce rAAV, which is harvested from the cells and purified. Production methods for rAAV involve delivery of three necessary elements to the producer cells: 1) a gene of interest flanked by AAV ITR sequences, 2) AAV rep and cap genes, and 3) helper virus proteins (“helper functions”). The conventional protocol for delivering the first two is by transfection of the cells with plasmid DNA containing the appropriate recombinant gene cassettes The helper functions have traditionally been supplied by infecting the cells with a helper virus such as adenovirus (Ad). (Samulski et al., 1998; Hauswirth et al., 2000).
1.3 Quantitative Problems Associated With Producing Amounts of rAAV Needed for Gene Therapy
Despite the potential benefits of gene therapy as a treatment for human genetic diseases, unfortunately, a serious practical limitation stands in the way of its widespread use in the clinic. In order to produce even a single clinically effective dose for a human patient, over 1014 rAAV particles must be made (Snyder, et al., 1997; Ye et al., 1999). To make this number of particles using current technology requires over 2×1011 producer cells. On a laboratory scale, this number of cells would require about 7500 tissue culture flasks. On a commercial scale, this level of cell culture poses a serious practical barrier to large scale production of rAAV in “cell factories.”
The benefits of improving particle yield per cell will be very significant from a commercial production standpoint. For example, an improvement resulting in a two-fold increase in rAAV yield per cell would allow for culture of half as many cells. A ten-fold increase would enable the same amount of rAAV product to be made by one-tenth the number of producer cells. Significant improvements of this magnitude are required in order to achieve economic feasibility for this technology.
Current rAAV production methodologies make use of procedures known to limit the number of rAAV that a single producer cell can make. The first of these is transfection using plasmids for delivery of DNA to the cells. It is well known that plasmid transfection is an inherently inefficient process requiring high genome copies and therefore large amounts of DNA (Hauswirth et al., 2000). Additionally, use of Ad significantly reduces the final rAAV titers because it is a contaminant that must be removed from the final product. Not only must effective procedures be employed to eliminate Ad contamination, but stringent assays for Ad contamination of rAAV are also necessary. Purification and safety procedures dictated by the use of Ad result in loss of rAAV at each step. To overcome the major barrier to the routine use of gene therapy, commercially practical methods must be developed to provide rAAV in the vast amounts required for clinical applications.